HIV Gag directs the process of particle assembly. Individual steps in HIV assembly are being defined, yet significant gaps in our knowledge remain. The identification of cellular factors interacting with Gag has been essential to recent advances in the field. In particular, the finding that Gag directly interacts with ESCRT components in the cell to achieve the late stages of budding provided a connection between vesicular transport pathways and Gag trafficking. Particle assembly occurs in a late endosomal compartment identified as the multivesicular body (MVB) in many cell types, and Gag can be found in MVBs even in cell types that demonstrate dominant plasma membrane budding. Our laboratory recently identified a novel interaction of Gag with the AP-3 transport complex, which plays a role in the trafficking of Gag to the MVB. A detailed dissection of the role of AP-3 in Gag trafficking and particle assembly is now warranted. The identification of the AP-3 trafficking step should facilitate the identification of additional discrete steps in Gag trafficking in the cell. The major hypothesis of this competing renewal application is that Gag reaches the MVB and subsequently the plasma membrane through discrete, sequential interactions with cellular transport pathways. Furthermore, we propose that differences in the abundance and intracellular location of specific cellular factors involved in HIV budding determine MVB vs plasma membrane assembly in different cell types. The overall goasl of the research plans are to define the discrete trafficking steps utilized by Gag and dissect the sequence of interactions of Gag with specific cellular factors involved in budding. A major emphasis will be on building on our recent finding of the Gag-AP-3 interaction to provide a better understanding of this early step and of subsequent events in assembly. Experiments in Aim 1 will define the biochemical an structural basis of the Gag-AP-3 delta subunit interaction. Aim II will define the sequential steps involved in the intracellular trafficking of Gag. Aim III will examine the pathway taken by Gag in moving from the MVB to the plasma membrane, focusing on the secretory lysosome pathway and effector molecules acting on this pathway. Together, these experiments will provide important new insights into the interaction of HIV structural proteins with cellular transport pathways.